Extrinsic Amyloid-Binding Dyes for the Detection of Individual Protein Aggregates in Solution

Protein aggregation is a key molecular feature underlying a wide array of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. To understand protein aggregation in molecular detail it is crucial to be able to characterise the array of heterogeneous aggregates that are formed during the aggregation process. We present here a high-throughput method to detect single protein aggregates, in solution, from a label-free aggregation reaction and demonstrate the approach with the protein associated with Parkinson's disease, alpha synuclein. The method combines single-molecule confocal microscopy with a range of amyloid-binding extrinsic dyes, including thioflavin T and pentameric formyl thiophene acetic acid, and we show that we can observe aggregates at low picomolar concentrations. The detection of individual aggregates allows us to quantify their numbers. Furthermore, we show that this approach also allows us to gain structural insights from the emission intensity of the extrinsic dyes that are bound to aggregates. By analysing the time evolution of the aggregate populations on a single-molecule level, we then estimate the fragmentation rate of aggregates, a key process which underlies the multiplication of pathological aggregates. We additionally demonstrate that the method permits the detection of these aggregates in biological samples. The capability to detect individual protein aggregates in solution opens up a range of new applications, including exploiting the potential of this method for high-throughput screening of human biofluids for disease diagnosis and early detection.ERC and Royal societ